This project aims to assess the role of caspases in T cell death generally, and in particular to identify the cytoplasmic caspase substrates whose cleavage gives rise to cell death. We have found caspase-dependent increases in the rate endocytosis in apoptotic cells of the human Jurkat T cell line and in mouse thymocytes in short term culture. These increases were triggered by a variety of different apoptotic stimuli in each case, and were specifically blockable by peptide-FMK caspase inhibitors, leading us to conclude that increased endocytosis is a new and previously unknown aspect of apoptosis. A molecular hypothesis to explain this increase is that caspases may cleave a regulatory protein normally restraining membrane fusion. One such protein is the syntaxin-binding protein munc-18, whose sequence shows two potential recognition sites for caspase 6. In vitro translated recombinant munc-18 was shown to be digested by recombinant caspase 6 into two predicted fragments indicating cleavage occurred at both sites. This munc-18 cleavage was blocked by the specific caspase 6 inhibitor VEID-CHO. Using an anti-munc-18 Mab to detect this protein in cell extracts by Western blotting, we found that one of the munc-18 fragments produced by caspase-6 appeared in anti-Fas-treated Jurkat cells and dexamethasone-treated thymocytes. Since munc-18 is known to inhibit exocytosis, we examined the rate of exocytosis of 125I-labeled transferring in apoptotic Jurkat cells. We found that anti-Fas treatment induced a ~4x increase in the rate of exocytosis, as predicted by the munc-18 cleavage model. To definitively test whether munc-18 cleavage causes apoptotic increases in vesicle trafficking, we are currently assessing this in cells overexpressing munc-18 mutated at the cleavage sites.We have also studied caspase activation in intact apoptotic cells using a novel class of cell-permeable fluorogenic caspase substrates. These substituted peptides were synthesized to contain the optimal tetrapeptide recognition motifs for caspases 1,3/7,6,8 and 9. Examination of thymocytes by flow cytometry showed that for each substrate a discrete brightly fluorescent cell population appeared starting 2-3 hours after dexamethasone addition, and the proportion of these bright cells increased over the next 5 hours. The sequential order of increase of caspase activities was LEHDase, WEHDase, VEIDase, IETDase and DEVDase, largely corresponding to the caspase cascade predicted from studies on apoptotic extracts. When thymocytes were treated with anti-Fas antibody, the order of caspase activities was clearly different, with IETDase appearing first, and DEVDase preceding VEIDase, Thus fluorogenic intracellullar caspase substrates allow direct observations of the caspase cascade in apoptotic cells and complement traditional biochemical techniques.